5-HT2 receptor binding sites are not up-regulated following denervation of serotonin terminals by a selective neurotoxin, suggesting that endogenous substance other than 5-HT exist for this receptor sites. We have, therefore, attempted to identify and isolate endogenous ligands for the 5-HT2 recognition site in the CNS. When 0.1 M acetic acid extract of bovine brain is applied a CM-Sephadex column, some fractions display activity of inhibiting 3H-ketanserin binding to the rat brain membranes. This activity is diminished by treatment with trypsin or pronase, suggesting that the material is protein in nature. This polypeptide has a molecular weight of about 6,000 daltons and inhibits the specific binding of 3H-Ketanserin and, to a slightly lesser extent 3H-mianserin, but fails to affect the binding of 3H-imipramine and 3H-dihydroalprenolol. This peptide has been partially purified by carboxylmethyl-sephardex chromatography, Biogel P-10 permeation chromatography and a reversed phase HPLC-C8 column chromatography. We have also tested alternative extraction procedures using 0.1 M HCl and various concentractions of organic solvent (such as accetonitrile) in an effort to maximize the recovery and to facilitate the purification to homogeneity of this protein. We are currently testing the biological activity of this polypeptde on 5-HT2 receptors by ascertaining its effects on (1) 5-HT sensitive adenylate cyclase activity in NCB-20 cells, (2) 5-HT stimulated phosphoinositide hydrolysis in brain slices and aorta of rats and (3) 5-HT-induced contraction of the ring of rat aorta. Since 5-HT2 receptor recognition sites are down-regulated following subchronic treatment of experimental animals with a variety of antidepressants, this putative peptide may be involved in this affective disorder and might be used as a biological marker for this mental illness.